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Immunohistochemical study on the distribution of the voltage-gated potassium channels in the gerbil hippocampus
Neuroscience Letters 298 (2001) 29±32
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Immunohistochemical study on the distribution of the voltagegated potassium channels in the gerbil hippocampus Kyeong Han Park a, Yoon Hee Chung a, Chung-Min Shin a, Myeung Ju Kim a, Byung Kwon Lee a, Sa Sun Cho a,b, Choong Ik Cha a,b,c,* a
Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea b Neuroscience Research Institute, Medical Research Center, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea c Biomedical Research Center, KNIH, Seoul, South Korea Received 16 October 2000; received in revised form 22 November 2000; accepted 22 November 2000
Abstract The differential expression of specialized voltage-gated potassium (Kv) channel subtypes probably re¯ects the wide range of functions in the nervous system. In the present study, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry. Immunoreactivities for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 were observed in the pyramidal cells of the CA1±CA3 areas. In addition, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4. Although there was some overlap, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies of the pyramidal cells and granule cells. As a whole, the present study has clearly shown Kv1 channel distributions in the gerbil hippocampus, which were variant between species and therefore more or less functionally signi®cant. This study may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kv channels; Gerbil; Hippocampus; CA1; CA3; Immunohistochemistry
The differential and neuronal cell type-speci®c expression of specialized voltage-gated potassium (Kv) channel subtypes in the nervous system may re¯ect the wide range of functions which Kv channels may exert as key determinants of membrane excitability. The Kv channels consist of a and b subunits [10]. Kv channel a subunits belong to a superfamily on the basis of structural relatedness [4]. Most likely, different combinations of Kv channel a and b subunit isoforms into heteromultimeric Kv channels may substantially contribute to the generation of Kv channel diversity in the nervous system [10]. Mutations in certain Kv channels have been associated with hyperexcitable phenotypes in both humans and animals [11,15,19]. Using behavioral and immediate-early gene indicators of regional brain excitability, Rho et al. [11] have found that a seizure-sensitive predisposition exists in Kv1.1 2/2 animals at a very young age. Smart et al. [15] * Corresponding author. Tel.: 182-2-740-8205; fax: 182-2-7459528. E-mail address: [email protected] (C.I. Cha).
have suggested that loss of Kv1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Madeja et al. [7] provided the evidence that the differences in distributions and properties of Kv channels contribute to differences in the seizure susceptibility of brain areas. Transient cerebral ischemia initiates a process of cellular events that lead to the delayed neuronal degeneration of several brain regions in animal models [5,9]. One of the most vulnerable neuronal populations resides in the CA1 region of hippocampus, a brain structure that is important for learning, memory and cognitive function [20]. Electrophysiological studies have indicated that the excitability of CA1 neurons is depressed following ischemia [17]. The diminished excitability, therefore, may be involved in the mechanisms of neuronal loss following ischemia [3]. It has been shown that the decreased excitability in CA1 neurons
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 0- 9
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K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
Fig. 1. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E) and Kv1.6 (F) channels in the gerbil hippocampus. The pyramidal cell bodies of CA1±3 areas and the granule cell bodies of the dentate gyrus highly expressed Kv1.1 (A), Kv1.4 (D) and Kv1.6 (F) channels. A distinct immunoreactivity pattern of Kv1.2 was observed in the middle third of the dentate gyrus molecular layer (B). CA1±3, ®elds CA1±3 of Ammon's horn; DG, dentate gyrus. Scale bar = 250 mm (A±F).
during hypoxia in vitro is mainly due to an increase in potassium conductance [6]. The aim of this study was to investigate differences in the spatial patterning of Kv1 channels in the gerbil hippocampus, which is among the most vulnerable to ischemia and epilepsy in the nervous system. Previous immunohistochemical studies of Kv channels in the rat and mouse brain revealed that some of them are segregated to speci®c subcellular regions [2,12,14,18]. However, there are no reports about Kv channel distribution in the gerbil, which is used as an ischemia and epilepsy animal model. Therefore, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry for the ®rst time. Surprisingly, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies. Five adult (8±10 week old) Mongolian gerbils (Meriones unguicularis) were used in this experiment. These animals were treated in accordance with the `Principles of Laboratory Animal Care' (NIH publication No. 86±23, revised in 1985). The animals were perfused transcardially with cold phosphate buffered saline (PBS, 0.02 M, pH 7.4), and then with ice-cold 4% paraformaldehyde for 10 min at a ¯ow rate of 50±60 ml/min. Brains were sliced into blocks 4±6 mm thick, immersed in a cold ®xative for 6±12 h, and then cryoprotected in a series of cold sucrose solutions of increasing concentration. Frozen sections were cut at 40 mm in the coronal plane. Immunohistochemistry was performed in accordance with the free-¯oating method described earlier [1]. Polyclonal anti-Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6 (product No. APC-009, 010, 002,
007, 004 and 003, Alomone Labs, Jerusalem, Israel) were used as primary antibodies with a dilution ratio of 1:200, 1:200, 1:70, 1:200, 1:70 and 1:70, respectively. A sample of sections was reacted without primary antiserum, and a different sample was exposed to a primary antiserum that had been preincubated for 24 h with control antigen peptides. Sections from these samples did not exhibit any of the immunoreactivity described in this report. Speci®city of immunoreactivity to six Kv1 channels in the gerbil brain was already con®rmed by immunohistochemistry in the rat brain [1]. The antibodies used here displayed a speci®c reaction to the a-subunits of Kv1 channels in the rat brain including the hippocampus. In the present study, each Kv channel subunit had a unique pattern of distribution in the hippocampus (Fig. 1). The pyramidal cell bodies of CA1±3 areas and the granule cell bodies of the dentate gyrus were strongly immunoreactive for Kv1.1, Kv1.4 and Kv1.6 (Fig. 1A,D,F). Although the staining intensity was relatively low, Kv1.2 was also expressed in these cell bodies (Fig. 1B). On the other hand, Kv1.3 and Kv1.5 subunits did not show any immunoreactivity in the pyramidal layer and granule cell layer (Fig. 1C,E). In particular, Kv 1.5 showed the cloud-like neuropil staining throughout the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus (Fig. 1E). In the gerbil hippocampus, the most intense staining for Kv1.1 channel was observed in the pyramidal cells of the CA3 region, with heavy labeling of granule cells in the dentate gyrus (Fig. 1A). Moderate staining was also observed in the pyramidal cells of the CA1 region. At a higher magni®cation, immunoreactivity in the pyramidal cells and the granule cells was seen in the cell bodies, but did not extend into the dendrites (Figs. 2A, 3A). Kv1.1immunoreactive cells were also scattered in other layers,
Fig. 2. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.4 (C) and Kv1.6 (D) channels in the dentate gyrus. Kv1.1 staining was intense in the granule cell layer, whereas Kv1.2 and Kv1.4 staining was moderate in the same layer. In addition to the different intensity, the staining pattern was also different among these four subunits. G, granular layer; M, molecular layer; Po, polymorphic layer. Scale bar = 50 mm (A±D).
K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
Fig. 3. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.4 (C) and Kv1.6 (D) channels in the CA3 region of the hippocampus. Immunoreactivity for these subunits was observed in the pyramidal cell bodies, with relatively low signals in the stratum oriens and stratum lucidum. The staining intensity for Kv1.4 was most intense among these subunits. L, stratum lucidum; O, stratum oriens; P, pyramidal cell layer. Scale bar = 50 mm (A±D).
such as the stratum oriens and stratum radiatum. A distinct immunoreactivity pattern of Kv1.2 was observed in the middle third of the dentate gyrus molecular layer, indicating a localization of Kv1.2 proteins in granule cell dendrites (Fig. 1B). Relatively weak labeling for Kv1.2 was observed in the granule cells in the dentate gyrus (Fig. 2B) and pyramidal cells of the CA1±3 areas (Fig. 3B). In the polymorphic layer of the dentate gyrus, there were large Kv1.2-immunoreactive cells (Fig. 2B). Expression of Kv1.4 channel proteins was observed in the pyramidal cells of the CA1±3 regions and granule cells of the dentate gyrus (Fig. 1D). It was noted that the most intense staining was found in the pyramidal layer of the CA3 region (Figs. 1D, 3C). Like the staining pattern of Kv1.2, large Kv1.4- and Kv1.6-immunoreactive cells were also in the polymorphic layer of the dentate gyrus (Fig. 2C,D). Interestingly, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4 (Fig. 4). Like other Kv1 subunits, Kv1.6 was expressed at a slightly higher level in the pyramidal cells of CA3 region than in the granule cells of the dentate gyrus (Figs. 1F, 2D, 3D). Studies on regional localization patterns of Kv1 channel subunits should provide helpful guidelines for correlating current types with particular channels. In this study, therefore, we described the regional localization of six members of Kv channel subunits in the gerbil hippocampus, which has been known to be closely associated with ischemia and epilepsy. In the present study, there were signi®cant differences from the previous studies described in the rat and mouse brain, in that we observed intense staining mainly in the pyramidal cells and granule cells. In the dentate gyrus of the rat, Kv1.1, Kv1.2 and Kv1.4 subunits were concentrated in a prominent band located in
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the middle third of the molecular layer [2,12,14,18]. As described previously, this pattern closely matches the termination zone of the medial perforant path. In inner and outer thirds of the molecular layer of the dentate gyrus, there was also diffuse immunoreactivity for Kv1.2, Kv1.4 and Kv1.6. They have suggested that this staining may represent subunit expression in the dendrites of dentate granule cells or in axons of other afferent inputs to the dentate gyrus. In the present study, an intense and well-de®ned band of Kv1.2 immunoreactivity occupied in the middle third of the molecular layer. On the other hand, we found immunoreactivity for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 in the dentate granule cells. In the CA3 region of the rat, there was a prominent band of immunoreactivity for Kv1.1 and Kv1.4 in a narrow zone immediately above pyramidal cell layer of CA3 [2,12,14], suggesting these subunits are associated with mossy ®ber axons [18]. In the gerbil CA3 sub®eld, however, there was not a clear band of immunoreactivity for these subunits. Immunoreactivity for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 was observed in the pyramidal cells of the CA1±CA3 areas, although the staining intensity seemed to be higher in the CA3 region. Interestingly, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4 and were also immunoreactive for Kv1.1, Kv.1.2 and Kv1.6. In particular, Kv1.1 protein distribution was in agreement with in situ hybridization in the mouse hippocampus, in which Kv1.1 mRNA was highly expressed in CA3 pyramidal cells [18]. Recently, Smart et al. [15] have reported that homozygous Kv1.1 null mice display frequent spontaneous seizures, correlating on the cellular level with alterations in hippocampal excitability and nerve conduction. These data have indicated that loss of Kv1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Therefore, our results have suggested that Kv1.1 channel concentrated in the CA3 region may be associated with epileptic activity in the gerbil.
Fig. 4. Distinct localization of Kv1.4 channel subunit in the stratum oriens of the hippocampus. Many medium- to large-sized interneurons located within stratum oriens of CA1±CA3 were strongly immunoreactive for Kv1.4. O, stratum oriens; P, pyramidal cell layer. Scale bar = 50 mm (A, B).
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K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
In the previous studies on the Kv1.4 subunit [2,14], the absence of cell body staining was a prominent feature of Kv1.4 immunocytochemistry in the rat hippocampus. Ultrastructural studies revealed that Kv1.4 immunoreactivity was absent from the surface membranes of cell bodies and dendrites and occurred prominently on axons [2]. The results of Sheng et al. [14] was quite different from our results in that we observed intense staining in the pyramidal cells and granule cells. Recently, the activity of Kv1.4 channels has been shown to highly sensitive to external K 1 concentration [8] and to reducing and oxidizing agents [13]. If the redox potential or the surrounding K 1 concentration changed signi®cantly during prolonged neuronal activity, then these mechanisms could provide a means by which Kv1.4 channels in axons and terminals are locally modulated in a use-dependent fashion. Traditionally ischemic neuronal death is considered to be a consequence of necrosis. However, in recent years, accumulating evidence has indicated that many neurons undergo apoptosis after global or focal ischemia. Studies have shown that increased K 1 ef¯ux might be a primary step leading to apoptosis. In epileptic research, it is assumed that a disturbance of Kv channel function is involved in epileptogenesis by leading to decreased neuronal stability. Recent studies have shown that potassium channel gene expression is altered in the hippocampus following seizure activity [16]. Therefore, this study on the differential distribution of Kv1 channels in the gerbil hippocampus may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy. This research was supported by Korean Ministry of Health and Welfare Research Foundation Grant HMP-98N-2-0019. This study was supported in part by year 2000 BK21 project for Medicine, Dentistry and Pharmacy. [1] Chung, Y.H., Shin, C., Kim, M.J. and Cha, C.I., Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat basal ganglia, Brain Res., 875 (2000) 164±170. [2] Cooper, E.C., Milroy, A., Jan, Y.N., Jan, L.Y. and Lowenstein, D.H., Presynaptic localization of Kv1.4-containing Atype potassium channels near excitatory synapses in the hippocampus, J. Neurosci., 18 (1998) 965±974. [3] Gao, T.M., Pulsinelli, W.A. and Xu, Z.C., Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience, 90 (1999) 771±780. [4] Gutman, G.A. and Chandy, K.G., Nomenclature for mammalian voltage-dependent potassium channel genes, The Neurosciences, 5 (1993) 101±104.
[5] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57±69. [6] Leblond, J. and Krnjevic, K., Hypoxic changes in hippocampal neurons, J. Neurophysiol., 62 (1989) 1±14. [7] Madeja, M., Musshoff, U. and Speckmann, E.J., Diversity of potassium channels contributing to differences in brain area-speci®c seizure susceptibility: sensitivity of different potassium channels to the epileptogenic agent pentylenetetrazol, Eur. J. Neurosci., 9 (1997) 390±395. [8] Pardo, L.A., Heinemann, S.H., Terlau, H., Ludewig, U., Lorra, C., Pongs, O., and Stuhmer, W., Extracellular K 1 speci®cally modulates a rat brain K 1 channel, Proc. Natl. Acad. Sci. USA, 89 (1992) 2466±2470. [9] Petito, C.K. and Pulsinelli, W.A., Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes, J. Cereb. Blood Flow Metab., 4 (1984) 194±205. [10] Rettig, J., Heinemann, S.H., Wunder, F., Lorra, C., Parcej, D.N., Dolly, J.O. and Pongs, O., Inactivation properties of voltage-gated K 1 channels altered by presence of betasubunit, Nature, 369 (1994) 289±294. [11] Rho, J.M., Szot, P., Tempel, B.L. and Schwartzkroin, P.A., Developmental seizure susceptibility of kv1.1 potassium channel knockout mice, Dev. Neurosci., 21 (1999) 320±327. [12] Rhodes, K.J., Keilbaugh, S.A., Barrezueta, N.X., Lopez, K.L. and Trimmer, J.S., Association and colocalization of K 1 channel a- and b-subunit polypeptides in rat brain, J. Neurosci., 15 (1995) 5360±5371. [13] Ruppersberg, J.P., Stocker, M., Pongs, O., Heinemann, S.H., Frank, R. and Koenen, M., Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation, Nature, 352 (1991) 711±714. [14] Sheng, M., Tsaur, M.L., Jan, Y.N. and Jan, L.J., Subcellular segregation of two A-type K 1 channel proteins in rat central neurons, Neuron, 9 (1992) 271±284. [15] Smart, S.L., Lopantsev, V., Zhang, C.L., Robbins, C.A., Wang, H., Chiu, S.Y., Schwartzkroin, P.A., Messing, A. and Tempel, B.L., Deletion of the K(V)1.1 potassium channel causes epilepsy in mice, Neuron, 20 (1998) 809±819. [16] Tsaur, M.L., Sheng, M., Lowenstein, D.H., Jan, Y.N. and Jan, L.Y., Differential expression of K 1 channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures, Neuron, 8 (1992) 1055±1067. [17] Urban, L., Neill, K.H., Crain, B.J., Nadler, J.V. and Somjen, G.G., Postischemic synaptic physiology in area CA1 of the gerbil hippocampus studied in vitro, J. Neurosci., 9 (1989) 3966±3975. [18] Wang, H., Kunkel, D.D., Schwartzkroin, P.A. and Tempel, B.L., Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain, J. Neurosci., 14 (1994) 4588±4599. [19] Zerr, P., Adelman, J.P. and Maylie, J., Characterization of three episodic ataxia mutations in the human Kv1.1 potassium channel, FEBS Lett., 431 (1998) 461±464. [20] Zola Morgan, S., Squire, L.R., Rempel, N.L., Clower, R.P. and Amaral, D.G., Enduring memory impairment in monkeys after ischemic damage to the hippocampus, J. Neurosci., 7 (1992) 2582±2596.
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Immunohistochemical study on the distribution of the voltagegated potassium channels in the gerbil hippocampus Kyeong Han Park a, Yoon Hee Chung a, Chung-Min Shin a, Myeung Ju Kim a, Byung Kwon Lee a, Sa Sun Cho a,b, Choong Ik Cha a,b,c,* a
Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea b Neuroscience Research Institute, Medical Research Center, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea c Biomedical Research Center, KNIH, Seoul, South Korea Received 16 October 2000; received in revised form 22 November 2000; accepted 22 November 2000
Abstract The differential expression of specialized voltage-gated potassium (Kv) channel subtypes probably re¯ects the wide range of functions in the nervous system. In the present study, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry. Immunoreactivities for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 were observed in the pyramidal cells of the CA1±CA3 areas. In addition, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4. Although there was some overlap, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies of the pyramidal cells and granule cells. As a whole, the present study has clearly shown Kv1 channel distributions in the gerbil hippocampus, which were variant between species and therefore more or less functionally signi®cant. This study may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kv channels; Gerbil; Hippocampus; CA1; CA3; Immunohistochemistry
The differential and neuronal cell type-speci®c expression of specialized voltage-gated potassium (Kv) channel subtypes in the nervous system may re¯ect the wide range of functions which Kv channels may exert as key determinants of membrane excitability. The Kv channels consist of a and b subunits [10]. Kv channel a subunits belong to a superfamily on the basis of structural relatedness [4]. Most likely, different combinations of Kv channel a and b subunit isoforms into heteromultimeric Kv channels may substantially contribute to the generation of Kv channel diversity in the nervous system [10]. Mutations in certain Kv channels have been associated with hyperexcitable phenotypes in both humans and animals [11,15,19]. Using behavioral and immediate-early gene indicators of regional brain excitability, Rho et al. [11] have found that a seizure-sensitive predisposition exists in Kv1.1 2/2 animals at a very young age. Smart et al. [15] * Corresponding author. Tel.: 182-2-740-8205; fax: 182-2-7459528. E-mail address: [email protected] (C.I. Cha).
have suggested that loss of Kv1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Madeja et al. [7] provided the evidence that the differences in distributions and properties of Kv channels contribute to differences in the seizure susceptibility of brain areas. Transient cerebral ischemia initiates a process of cellular events that lead to the delayed neuronal degeneration of several brain regions in animal models [5,9]. One of the most vulnerable neuronal populations resides in the CA1 region of hippocampus, a brain structure that is important for learning, memory and cognitive function [20]. Electrophysiological studies have indicated that the excitability of CA1 neurons is depressed following ischemia [17]. The diminished excitability, therefore, may be involved in the mechanisms of neuronal loss following ischemia [3]. It has been shown that the decreased excitability in CA1 neurons
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 0- 9
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K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
Fig. 1. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E) and Kv1.6 (F) channels in the gerbil hippocampus. The pyramidal cell bodies of CA1±3 areas and the granule cell bodies of the dentate gyrus highly expressed Kv1.1 (A), Kv1.4 (D) and Kv1.6 (F) channels. A distinct immunoreactivity pattern of Kv1.2 was observed in the middle third of the dentate gyrus molecular layer (B). CA1±3, ®elds CA1±3 of Ammon's horn; DG, dentate gyrus. Scale bar = 250 mm (A±F).
during hypoxia in vitro is mainly due to an increase in potassium conductance [6]. The aim of this study was to investigate differences in the spatial patterning of Kv1 channels in the gerbil hippocampus, which is among the most vulnerable to ischemia and epilepsy in the nervous system. Previous immunohistochemical studies of Kv channels in the rat and mouse brain revealed that some of them are segregated to speci®c subcellular regions [2,12,14,18]. However, there are no reports about Kv channel distribution in the gerbil, which is used as an ischemia and epilepsy animal model. Therefore, we investigated the distribution of six Kv1 channel subtypes in the gerbil hippocampus by immunohistochemistry for the ®rst time. Surprisingly, our results were quite different from the previous studies described in the rat and mouse hippocampus, in that we observed intense staining mainly in the cell bodies. Five adult (8±10 week old) Mongolian gerbils (Meriones unguicularis) were used in this experiment. These animals were treated in accordance with the `Principles of Laboratory Animal Care' (NIH publication No. 86±23, revised in 1985). The animals were perfused transcardially with cold phosphate buffered saline (PBS, 0.02 M, pH 7.4), and then with ice-cold 4% paraformaldehyde for 10 min at a ¯ow rate of 50±60 ml/min. Brains were sliced into blocks 4±6 mm thick, immersed in a cold ®xative for 6±12 h, and then cryoprotected in a series of cold sucrose solutions of increasing concentration. Frozen sections were cut at 40 mm in the coronal plane. Immunohistochemistry was performed in accordance with the free-¯oating method described earlier [1]. Polyclonal anti-Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6 (product No. APC-009, 010, 002,
007, 004 and 003, Alomone Labs, Jerusalem, Israel) were used as primary antibodies with a dilution ratio of 1:200, 1:200, 1:70, 1:200, 1:70 and 1:70, respectively. A sample of sections was reacted without primary antiserum, and a different sample was exposed to a primary antiserum that had been preincubated for 24 h with control antigen peptides. Sections from these samples did not exhibit any of the immunoreactivity described in this report. Speci®city of immunoreactivity to six Kv1 channels in the gerbil brain was already con®rmed by immunohistochemistry in the rat brain [1]. The antibodies used here displayed a speci®c reaction to the a-subunits of Kv1 channels in the rat brain including the hippocampus. In the present study, each Kv channel subunit had a unique pattern of distribution in the hippocampus (Fig. 1). The pyramidal cell bodies of CA1±3 areas and the granule cell bodies of the dentate gyrus were strongly immunoreactive for Kv1.1, Kv1.4 and Kv1.6 (Fig. 1A,D,F). Although the staining intensity was relatively low, Kv1.2 was also expressed in these cell bodies (Fig. 1B). On the other hand, Kv1.3 and Kv1.5 subunits did not show any immunoreactivity in the pyramidal layer and granule cell layer (Fig. 1C,E). In particular, Kv 1.5 showed the cloud-like neuropil staining throughout the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus (Fig. 1E). In the gerbil hippocampus, the most intense staining for Kv1.1 channel was observed in the pyramidal cells of the CA3 region, with heavy labeling of granule cells in the dentate gyrus (Fig. 1A). Moderate staining was also observed in the pyramidal cells of the CA1 region. At a higher magni®cation, immunoreactivity in the pyramidal cells and the granule cells was seen in the cell bodies, but did not extend into the dendrites (Figs. 2A, 3A). Kv1.1immunoreactive cells were also scattered in other layers,
Fig. 2. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.4 (C) and Kv1.6 (D) channels in the dentate gyrus. Kv1.1 staining was intense in the granule cell layer, whereas Kv1.2 and Kv1.4 staining was moderate in the same layer. In addition to the different intensity, the staining pattern was also different among these four subunits. G, granular layer; M, molecular layer; Po, polymorphic layer. Scale bar = 50 mm (A±D).
K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
Fig. 3. Distinct localizations of Kv1.1 (A), Kv1.2 (B), Kv1.4 (C) and Kv1.6 (D) channels in the CA3 region of the hippocampus. Immunoreactivity for these subunits was observed in the pyramidal cell bodies, with relatively low signals in the stratum oriens and stratum lucidum. The staining intensity for Kv1.4 was most intense among these subunits. L, stratum lucidum; O, stratum oriens; P, pyramidal cell layer. Scale bar = 50 mm (A±D).
such as the stratum oriens and stratum radiatum. A distinct immunoreactivity pattern of Kv1.2 was observed in the middle third of the dentate gyrus molecular layer, indicating a localization of Kv1.2 proteins in granule cell dendrites (Fig. 1B). Relatively weak labeling for Kv1.2 was observed in the granule cells in the dentate gyrus (Fig. 2B) and pyramidal cells of the CA1±3 areas (Fig. 3B). In the polymorphic layer of the dentate gyrus, there were large Kv1.2-immunoreactive cells (Fig. 2B). Expression of Kv1.4 channel proteins was observed in the pyramidal cells of the CA1±3 regions and granule cells of the dentate gyrus (Fig. 1D). It was noted that the most intense staining was found in the pyramidal layer of the CA3 region (Figs. 1D, 3C). Like the staining pattern of Kv1.2, large Kv1.4- and Kv1.6-immunoreactive cells were also in the polymorphic layer of the dentate gyrus (Fig. 2C,D). Interestingly, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4 (Fig. 4). Like other Kv1 subunits, Kv1.6 was expressed at a slightly higher level in the pyramidal cells of CA3 region than in the granule cells of the dentate gyrus (Figs. 1F, 2D, 3D). Studies on regional localization patterns of Kv1 channel subunits should provide helpful guidelines for correlating current types with particular channels. In this study, therefore, we described the regional localization of six members of Kv channel subunits in the gerbil hippocampus, which has been known to be closely associated with ischemia and epilepsy. In the present study, there were signi®cant differences from the previous studies described in the rat and mouse brain, in that we observed intense staining mainly in the pyramidal cells and granule cells. In the dentate gyrus of the rat, Kv1.1, Kv1.2 and Kv1.4 subunits were concentrated in a prominent band located in
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the middle third of the molecular layer [2,12,14,18]. As described previously, this pattern closely matches the termination zone of the medial perforant path. In inner and outer thirds of the molecular layer of the dentate gyrus, there was also diffuse immunoreactivity for Kv1.2, Kv1.4 and Kv1.6. They have suggested that this staining may represent subunit expression in the dendrites of dentate granule cells or in axons of other afferent inputs to the dentate gyrus. In the present study, an intense and well-de®ned band of Kv1.2 immunoreactivity occupied in the middle third of the molecular layer. On the other hand, we found immunoreactivity for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 in the dentate granule cells. In the CA3 region of the rat, there was a prominent band of immunoreactivity for Kv1.1 and Kv1.4 in a narrow zone immediately above pyramidal cell layer of CA3 [2,12,14], suggesting these subunits are associated with mossy ®ber axons [18]. In the gerbil CA3 sub®eld, however, there was not a clear band of immunoreactivity for these subunits. Immunoreactivity for Kv1.1, Kv1.2, Kv1.4 and Kv1.6 was observed in the pyramidal cells of the CA1±CA3 areas, although the staining intensity seemed to be higher in the CA3 region. Interestingly, many medium- to large-sized interneurons located within stratum pyramidals, stratum oriens, and stratum radiatum of CA1±CA3 were strongly immunoreactive for Kv1.4 and were also immunoreactive for Kv1.1, Kv.1.2 and Kv1.6. In particular, Kv1.1 protein distribution was in agreement with in situ hybridization in the mouse hippocampus, in which Kv1.1 mRNA was highly expressed in CA3 pyramidal cells [18]. Recently, Smart et al. [15] have reported that homozygous Kv1.1 null mice display frequent spontaneous seizures, correlating on the cellular level with alterations in hippocampal excitability and nerve conduction. These data have indicated that loss of Kv1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Therefore, our results have suggested that Kv1.1 channel concentrated in the CA3 region may be associated with epileptic activity in the gerbil.
Fig. 4. Distinct localization of Kv1.4 channel subunit in the stratum oriens of the hippocampus. Many medium- to large-sized interneurons located within stratum oriens of CA1±CA3 were strongly immunoreactive for Kv1.4. O, stratum oriens; P, pyramidal cell layer. Scale bar = 50 mm (A, B).
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K.H. Park et al. / Neuroscience Letters 298 (2001) 29±32
In the previous studies on the Kv1.4 subunit [2,14], the absence of cell body staining was a prominent feature of Kv1.4 immunocytochemistry in the rat hippocampus. Ultrastructural studies revealed that Kv1.4 immunoreactivity was absent from the surface membranes of cell bodies and dendrites and occurred prominently on axons [2]. The results of Sheng et al. [14] was quite different from our results in that we observed intense staining in the pyramidal cells and granule cells. Recently, the activity of Kv1.4 channels has been shown to highly sensitive to external K 1 concentration [8] and to reducing and oxidizing agents [13]. If the redox potential or the surrounding K 1 concentration changed signi®cantly during prolonged neuronal activity, then these mechanisms could provide a means by which Kv1.4 channels in axons and terminals are locally modulated in a use-dependent fashion. Traditionally ischemic neuronal death is considered to be a consequence of necrosis. However, in recent years, accumulating evidence has indicated that many neurons undergo apoptosis after global or focal ischemia. Studies have shown that increased K 1 ef¯ux might be a primary step leading to apoptosis. In epileptic research, it is assumed that a disturbance of Kv channel function is involved in epileptogenesis by leading to decreased neuronal stability. Recent studies have shown that potassium channel gene expression is altered in the hippocampus following seizure activity [16]. Therefore, this study on the differential distribution of Kv1 channels in the gerbil hippocampus may provide useful data for the future investigations on the pathological conditions such as ischemia and epilepsy. This research was supported by Korean Ministry of Health and Welfare Research Foundation Grant HMP-98N-2-0019. This study was supported in part by year 2000 BK21 project for Medicine, Dentistry and Pharmacy. [1] Chung, Y.H., Shin, C., Kim, M.J. and Cha, C.I., Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat basal ganglia, Brain Res., 875 (2000) 164±170. [2] Cooper, E.C., Milroy, A., Jan, Y.N., Jan, L.Y. and Lowenstein, D.H., Presynaptic localization of Kv1.4-containing Atype potassium channels near excitatory synapses in the hippocampus, J. Neurosci., 18 (1998) 965±974. [3] Gao, T.M., Pulsinelli, W.A. and Xu, Z.C., Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo, Neuroscience, 90 (1999) 771±780. [4] Gutman, G.A. and Chandy, K.G., Nomenclature for mammalian voltage-dependent potassium channel genes, The Neurosciences, 5 (1993) 101±104.
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